Divergent outcomes of anti-PD-L1 treatment coupled with host-intrinsic differences in TCR repertoire and distinct T cell activation states in responding versus non-responding tumors

Abstract

Differential responses to immune checkpoint inhibitors (ICI) may be attributed to tumor-intrinsic factors or environmental cues; however, these mechanisms cannot fully explain the variable ICI responses in different individuals. Here, we investigate the potential contribution of immunological heterogeneity with a focus on differences in T-cell receptor (TCR) repertoire to ICI responses, which has not been defined previously. To reveal additional factors underlying heterogeneous responses to ICI, we employed a squamous cell carcinoma (SCC) mouse model in which tumor-bearing recipients unambiguously diverged into responders (R) or non-responders (NR) upon anti-PD-L1 treatment. Treatment efficacy absolutely required CD8 T-cells and correlated positively with effector functions of CD8 tumor-infiltrating lymphocytes (TILs). We showed that TCR repertoires exhibited a similar magnitude of clonal expansion in R vs. NR CD8 TILs. However, the top expanded TCR clonotypes appeared to be mutually exclusive between R and NR CD8 TILs, which also occurred in a recipient-specific manner, demonstrating preferential expansion of distinct TCR clonotypes against the same SCC tumor. Unexpectedly, R vs. NR CD8 TILs reached all activation clusters and did not exhibit substantial global differences in transcriptomes. By linking single-cell transcriptomic data with unique TCR clonotypes, CD8 TILs harboring top TCR clonotypes were found to occupy distinct activation clusters and upregulate genes favoring anti-tumor immunity to different extents in R vs. NR. We conclude that stochastic differences in CD8 TIL TCR repertoire and distinct activation states of top TCR clonotypes may contribute to differential anti-PD-L1 responses. Our study suggests that host-intrinsic immunological heterogeneity may offer a new explanation for differential ICI responses in different individuals, which could impact on strategies for personalized cancer immunotherapy.

Host-specific differences in top-expanded TCR clonotypes correlate with divergent outcomes of anti-PD-L1 treatment in responders versus non-responders

“…We made a few unexpected discoveries (1): there were no differences in clonal expansion status or TCR diversity indexes between R vs. NR CD8 TILs (2); top expanded TCR clonotypes differed substantially, almost mutually exclusive, between R vs. NR CD8 TILs, when the threshold of clonal frequency was set as >1% or >0.5%. This observation is consistent with our previous single-cell sequencing results ( 19); (3) however, when clonotypes with lower frequencies were included, we failed to detect differences in TCR clonotypes between R vs. NR CD8 TILs, a finding not observed in our previous study (19). Basically, in the current study, we found that R clonotypes were also detected in NR recipients, albeit at a much lower frequency, and vice versa.…”

“…injection three times (2-day interval) or PBS only as vehicle control (control group). Of note, as shown in our previous study (19), all the tumor-bearing mice in the control group had tumor growing out, and no substantial differences were observed in tumor growth among control group. To assess treatment effects, relative change in tumor volume (RCTV) was calculated as the change in tumor volume (TV) from the start of treatment (TV 0 ) to the TV at day n (the endpoint of control group) (TV n ) divided by TV 0 (RCTV=[TV n −TV 0 ]/TV 0 ).…”

“…Prior studies showed that the level of CD8 tumor-infiltrating lymphocytes (TILs) correlate positively with HNSCC outcomes (15)(16)(17)(18). Given the critical role of CD8 T cells in anti-tumor immunity, we employed a mouse SCC model, where tumor-bearing recipients diverged into responders vs. non-responders upon anti-PD-L1 treatment, to examine the contribution of CD8 T cells to differential responses to ICI (19). In line with prior studies, we showed that CD8 T cells were required for the efficacy of anti-PD-L1 treatment.…”

“…In line with prior studies, we showed that CD8 T cells were required for the efficacy of anti-PD-L1 treatment. Moreover, we showed that top expanded TCR clonotypes were almost mutually exclusive in the CD8 TILs of responders vs. non-responders (19). We previously examined the TCR repertoire using single-cell TCR sequencing (19,20), which can simultaneously detect both TCRa and TCRb chains; however, the sequencing depth of this method is rather limited.…”

“…Moreover, we showed that top expanded TCR clonotypes were almost mutually exclusive in the CD8 TILs of responders vs. non-responders (19). We previously examined the TCR repertoire using single-cell TCR sequencing (19,20), which can simultaneously detect both TCRa and TCRb chains; however, the sequencing depth of this method is rather limited. Hence, in the current study, we employed the immunoSEQ platform to perform in-depth sequencing of TCRb clonotypes in the CD8 TILs of responders and non-responders.…”